Plasmid preparation problem using alkaline-lysis - (Jun/02/2005 )
Maybe it is a dumb problem. But for me, it is really a trouble.I use the alkaline-lysis method to extract the plasmids from E coli.. But the plasmid always not clear enough to do the digestion. What can I do? During the extraction, I use Phenol ( Tris saturated ), Phenol/chloroform and then chloroform again to clear the proteins. After that it is not clear enough.
another question:
When can I add the RNase to the solution? Since if i add it when i do the digestion, it may inhibit the activity of the Enzyme. Please give me your comments. Thanks.
hi. I also had same probem once, using the alkiline lysis protocol to extract my plasmid. I then used the boiling protocol and lysozyme my problem was solved, I could get very nice bands.
if boiling protocol doesn' work, maybe it is a problem of your the host E coli, some strain express some recombinases which can integrate the plasmid DNA with the genomic DNA, so you can never extract bac your plasmid.
if this is the problem in your case try to use E Coli DH5alpha. it works pretty well
good luck
When can I add the RNase to the solution? Since if i add it when i do the digestion, it may inhibit the activity of the Enzyme. Please give me your comments. Thanks.
Use Qiagen kit for plasmid isolation. I use it and don't have to bother about RNA contamination. With the boiling prep i got good bands too. If you want to know the protocol PM me.
another question:
When can I add the RNase to the solution? Since if i add it when i do the digestion, it may inhibit the activity of the Enzyme. Please give me your comments. Thanks.
They say AquaPlasmid does not use RNase. That may solve your problem. Good luck.
I had the same problem, particularly with those weak cutters like SmaI. You may want to try AquaPlasmid (http://www.aquaplasmid.com/). The 260/280 I got using AquaPlasmid was 2.1 and my SmaI cut finally works. It's really simple and doesn't use phenol or column or beads. I like it very much.
Thanks ! 
From your posts, I think maybe using the kits is the best way to solve the problem. Also, I will try the boil-lysis approach. I once used the boil-lysis, and i think the protocol is too much. So now i forget to use it again. Thanks.
Good Luck!
ning